Mitochondrion is a critical organelle present in most of the eukaryotic cells. Commonly known as the “power house of the cell” it is not only responsible for the production of ATP, but also generates superoxide ions and regulates cellular calcium levels and apoptotic cell death stimuli. Overall, it is involved in multiple cellular processes including Oxidative Phosphorylation, Apoptosis, Organizing Cell Signaling Pathways, and Regulation of Cell Cycle. Human Mitochondrial DNA (mtDNA) consists of 16,569 bp in a double-stranded circular structure and encodes 37 genes (13 respiratory genes, 2 rRNAs and 22 tRNAs).
FIG. 1 is a map of human mitochondrial DNA. Genes are transcribed as polycistronic RNA precursors that are later processed by RNAse P which recognizes the secondary structure of the tRNAs flanking each gene. The D-loop is non-coding and acts as a regulatory region, containing the H-chain and L-chain transcription initiation sites. Origins of replication are also shown.
mtDNA is used to study human migration and evolution. mtDNA is inherited from the mother, and does not undergo recombination, thereby providing a molecular clock to study evolutionary changes. Mutations in mtDNA are implicated in various diseases including metabolic disorders, neurodegenerative diseases, and cancer. More than 200 different mutations in the mtDNA have been associated with human diseases. Thus, sequencing the mitochondria can provide clues to origins of diseases as well as help delineate changes due to aging as well as somatic and germ-line mutations that might cause disorders. With the advent of next generation sequencing technology, it is now easy to deeply sequence DNA. In order to sequence the mtDNA, it is important to isolate the mtDNA effectively without any nuclear DNA contamination, because there are significant regions of homology between the nuclear and mitochondrial DNA, making it difficult to unambiguously identify the origin of short read fragments. Owing to the immense size difference between human nuclear DNA (6 billion bp) and human mtDNA (16 kb), small amounts of nuclear DNA contamination can lead to most of the DNA sequences being of nuclear origin. All current methods of isolating mitochondrial DNA involve first isolating the organelle and then purifying its DNA using standard protocols. These methods involving CsCl gradients, take advantage of the differential density of the circular mtDNA and the nuclear genomic DNA. The organelle separation leads to the loss of material, both due to the high forces involved (which can disrupt organelles and genomic DNA) and co-separation of the organelles with nuclear DNA due to similar densities. What is needed in the art are methods to efficiently and rapidly isolate mtDNA, while avoiding contamination with nuclear DNA.